This study will examine excitation-contraction coupling in cardiac ventricular muscle. The study will focus on (1) the calcium current, (2) the sodium-calcium exchange and (3) calcium release from the sarcoplasmic reticulum and their role in determining the rise in intracellular calcium that activates contraction in cardiac muscle. Enzymatically dissociated single cells will be voltage clamped with a single micro-electrode technique while free intracellular calcium will be measured using Fura-2 and/or Indo-1. Specific experiments will determine, for the first time, the voltage and time dependence of the intracellular calcium transient. The possible involvement of the calcium current in triggering the release of calcium from the sarcoplasmic reticulum will be examined in detail, to test the hypothesis that the calcium current provides the trigger for the calcium-induced release of calcium from the sarcoplasmic reticulum (Fabiato, 1985c). In addition, the possible involvement of the sodium-calcium exchange in supplying the calcium influx needed to activate the calcium-induced release of calcium from the sarcoplasmic reticulum will be examined. Experiments will also determine the relative contributions of the calcium current, the calcium release from the sarcoplasmic reticulum and calcium influx via the sodium-calcium exchange in the determination of the amplitude of the calcium transient. The dye signals will calibrated both in vitro and inside the cell to enable determination of free intracellular calcium levels from the dye signals. In addition, the kinetics of the dye reaction with calcium will be investigated to enable evaluation of the peak rate of calcium release from the sarcoplasmic reticulum. A computer model will be used to test hypotheses on how calcium release is regulated and the relative contributions of the calcium current, the sodium calcium exchange and sarcoplasmic reticulum release to the time course of the calcium transient. In addition, this model will also allow correction of the dye signals for the kinetics of the dye response as well as allowing estimation of the effects of possible dye exclusion from the myofilament space. This novel study will therefore provide a quantitative basis for understanding the regulation of intracellular calcium in cardiac muscle and the regulation of cardiac contractility. Such information should prove useful for the future development of new inotropic agents and provide a sound scientific basis for the understanding of cardiac failure.